LupiQuant: A real-time PCR based assay for determining host-to-parasite DNA ratios of Onchocerca lupi and host Canis lupus from onchocercosis samples

Abstract

Onchocerca lupi is a filarial nematode that causes ocular onchocercosis in canines globally including North America and areas of Europe, North Africa, and the Middle East. Reported incidence of this parasite in canines has continued to steadily escalate since the early 21st century and was more recently documented in humans. Whole genome sequencing (WGS) of this parasite can provide insight into gene content, provide novel surveillance targets, and elucidate the origin and range expansion. However, past attempts of whole genome sequencing of other Onchocerca species reported a substantial portion of their data unusable due to the variable over-abundance of host DNA in samples. Here, we have developed a method to determine the host-to-parasite DNA ratio using a quantitative PCR (qPCR) approach that relies on two standard plasmids each of which contains a single copy gene specific to the parasite genus Onchocerca (major body wall myosin gene, myosin) or a single copy gene specific to the canine host (polycystin-1 precursor, pkd1). These plasmid standards were used to determine the copy number of the myosin and pkd1 genes within a sample to calculate the ratio of parasite and host DNA. Furthermore, whole genome sequence (WGS) data for three O. lupi isolates were consistent with our host-to-parasite DNA ratio results. Our study demonstrates, despite unified DNA extraction methods, variable quantities of host DNA within any one sample which will likely affect downstream WGS applications. Our quantification assay of host-to-parasite genome copy number provides a robust and accurate method of assessing canine host DNA load in an O. lupi specimen that will allow informed sample selection for WGS. This study has also provided the first whole genome draft sequence for this species. This approach is also useful for future focused WGS studies of other parasites.

Biting midges (Diptera: Ceratopogonidae) as putative vectors of zoonotic Onchocerca lupi (Nematoda: Onchocercidae) in northern Arizona and New Mexico, southwestern United States

“…Current O. lupi surveillance tools targeting the COI mitochondrial gene are limited to detection and cannot provide insight into population structure, at least within the United States. Two recent studies published the first mitochondrial genome as well the first draft genome for O. lupi ( 18 , 63 ). These studies should facilitate the development of high-resolution surveillance tools for O. lupi to understand the genetic diversity and relationships among isolates globally.…”